Adhesion of cells to other cells and to the extracellular matrix is a fundamental characteristic of all multicellular organisms. Adhesions provide not only structural links between the intracellular cytoskeleton and the extracellular environment, but also provide sites of signal transduction that affect many aspects of cell behavior. This grant is aimed at understanding how signals from cell-matrix and cell-cell adhesions regulate members of the Rho family of GTPases, which are themselves key regulators of the cytoskeleton and many intracellular processes. The goal of the first aim is to determine how adhesion to the extracellular matrix protein fibronectin stimulates RhoA activity. The respective roles of integrins and syndecan-4 will be investigated. We will explore the contribution of specific integrin type, density of expression and clustering on RhoA activation. We will test the hypothesis that some of syndecan-4's effects are mediated through activation of Rap1, another low molecular weight GTPase. Strategies for identifying and isolating guanine nucleotide exchange factors (GEFs) involved in RhoA activation will be used. We will explore signaling pathways that regulate these GEFs. Cells can sense the physical state of the surface to which they adhere and we will test the hypothesis that isometric tension can stimulate RhoA activity. RhoA activity will be measured using biosensors expressed in single cells and live cell imaging. We will determine whether the application of tension to single cells locally activates RhoA. The second aim is directed in part toward identifying the Rac1 GEFs that are activated in response to cadherin engagement. In preliminary work, we have discovered that many Rho family GEFs bind to PDZ domains. Proteins with PDZ domains are typically enriched in cell-cell junctions. We will determine whether the binding of GEFs to PDZ domains serves to recruit them to cell junctions and whether this interaction regulates their activity.